Plague is an acute infection caused by Yersinia pestis, a Gram-negative, facultative intracellular bacterium that infects and survives in macrophages. Growing evidence suggests that intracellular growth is important for mammalian colonization by Y. pestis. Upon entry into macrophages, Y. pestis remains in a membrane-bound compartment called the Yersinia-containing vacuole (YCV), but it is not killed by the macrophage. Furthermore, the bacterium actively inhibits acidification of the YCV. We hypothesize that Y. pestis interacts with specific host factors in order to alter YCV maturation and acidification. While bacterial factors important for intracellular survival have been identifie, the molecular mechanisms used by the bacterium to survive within macrophages have not been defined. To identify these factors/pathways, we have built a novel Y. pestis bioreporter to monitor Y. pestis survival in macrophages. Using this bioreporter, we have developed an inhibitory RNA-based assay to specifically identify host factors that alter Y. pestis intracellular survival. With this assay we will perform a genome wide screen to identify host factors/pathways required for Y. pestis survival in macrophages (Aim 1). Using a combination of in silico and microscopy analysis, we will also specifically identify host factors identified in our screen that alter phagosome maturation to the benefit of Y. pestis (Aim 2). These studies will be the first to target the host cell to understand the mechanisms used by Y. pestis to survive in host cells.